Abstract

Inositol polyphosphate 5-phosphatase (5PTase) is a key enzyme in the phosphatidylinositol metabolic pathway, which plays critical roles in a number of cellular processes in plants. Our previous work implicated the role of 5PTase13, which encodes a WD40-containing type II 5PTase, in hormone-mediated cotyledon vein development. Here, we show that 5PTase13 is also involved in blue light responses in Arabidopsis thaliana. Compared with that in darkness, the expression of 5PTase13 was suppressed by blue light irradiation, and disruption of the gene resulted in shortened hypocotyls and expanded cotyledons. Genetic analysis showed that 5PTase13 acted independently from CRYPTOCHROME1 and CONSTITUTIVE PHOTOMORPHOGENIC1 but interacted functionally with PHOTOTROPIN1 (PHOT1). The expression level of 5PTase13 was significantly enhanced in phot1 single or phot1 phot2 double mutants under blue light, and suppression of 5PTase13 expression rescued the elongated hypocotyls in the phot1 or phot1 phot2 mutants. Further analysis showed that the blue light-induced elevation of cytosolic Ca2+ was inhibited in the phot1 mutant but enhanced in the 5pt13 mutant, suggesting that 5PTase13 antagonizes PHOT1-mediated effects on calcium signaling under blue light.

5PTase13 Functions Independently of COP1 and CRY1.(A) Yeast two-hybrid analysis showed no interaction between 5PTase13 and COP1 or CRY1. Interaction between the N-terminal region (until the position before WD40 region) or N-WD40 fragment (until the position at end of the WD40 region) of 5PTase13 with COP1 or CRY1 proteins was determined, respectively. The known interaction between pJG4-5-COP1 and pEG202-CCT1 (CRY1 C-terminal) was used as a positive control (P-K). The interaction between empty vectors pEG202 and pJG4-5 was used as the negative control (N-K).(B)cry1 5pt13 double mutants had an intermediate phenotype, indicating that there was no direct link between CRY1 and 5PTase13. Hypocotyl lengths were measured and data presented as described in .(C)cop1 5pt13 double mutants exhibited a cop1 phenotype, indicating that COP1 either acts downstream of 5PTase13 or they have no functional relationship. Hypocotyl lengths were measured and data presented as described in .

Deficiency of 5PTase13 Rescues the Shortened Hypocotyls of phot1.(A) Quantitative real-time RT-PCR analysis showed that 5PTase13 transcriptions were enhanced in phot1 and phot1 phot2 but not in phot2 seedlings under blue light (left panel; error bars represent se of three independent replicates; **, P < 0.01). Compared with elongated hypocotyls of phot1, phot2, and phot1 phot2 under blue light (20 μmol/m2/s), 5pt13 had shortened hypocotyls (middle panel). Relative hypocotyl lengths, compared with that of dark-grown seedlings, are shown in the right panel. See for procedure and data presentation.(B) Deficiency of 5PTase13 rescued the elongated hypocotyls of phot1 under blue light (20 μmol/m2/s; left panel). The lengths of hypocotyls were measured (middle panel), and the relative length was calculated (right panel).(C) Suppressed expression of 5PTase13 in phot1 as revealed by RT-PCR analysis (left panel) and correlated with shortened hypocotyls under blue light (20 μmol/m2/s; middle panel). The relative length is shown in the right panel.(D) RT-PCR analysis showing suppressed expression of 5PTase13 in phot1 phot2 (left panel) correlated with shortened hypocotyls under blue light (20 μmol/m2/s; middle panel). The relative length is shown in the right panel.

5PTase13 Overexpression Enhances Hypocotyl Elongation of phot1 but Not phot2.(A) Enhanced expression of 5PTase13 in phot1 (top panel) resulted in elongated hypocotyls compared with phot1 under blue light (bottom panel).(B) Seedlings with deficiency of 5PTase13 in the phot2 mutant background had similar hypocotyl length with phot2 (bottom panel), and overexpression of 5PTase13 in phot2 (top panel) did not result in shortened hypocotyl elongation under blue light (20 μmol/m2/s; bottom panel).

5PTase13 Deficiency Results in Increased [Ca2+]cyt and Renders Responses to Exogenous EGTA under Blue Light.(A) [Ca2+]cyt was increased in 5pt13 (left panel), consistent with the increased content of Ins(1,4,5)P3 in 5pt13 (right panel). The Ins(1,4,5)P3 content was reduced under overexpressed 5PTase13. Image analysis was performed by UV confocal imaging. Root hairs were submerged with 20 μM Indo-1 and pseudocolor according to the cytoplasmic calcium levels. The data were representative of root hairs from >10 individual roots. Five-day-old seedlings grown on Murashige and Skoog (MS) medium under white light were used to measure the Ins(1,4,5)P3 contents. Line 7 without overexpressed 5PTase13 after transformation (see Supplemental Figure 2A online) was used as a negative control. Assays were performed in triplicate, and the experiment was repeated two times and statistically analyzed using a one-tailed Student's t test (P < 0.01).(B)5pt13 had altered responses to exogenous EGTA under both high (top panel) and low (low panel) fluence rates of blue light. Hypocotyl lengths of 5-d-old seedlings were measured and statistically analyzed using a one-tailed Student's t test (*, P < 0.05; **, P < 0.01). Error bars represent se (n > 30).